AGATCCACTAGTTCTAGAGCTCAACTTGTTGGTTTGTCTTT TGTCCAAAAACGGTTTCCAG TGCCTTTCTGTTCGATTGAT

Transcription

1 1 Table S1. PCR primers Primer Use Sequence (5 3 ) JC17 SAT1 flipper GGCCCCCCCTCGAGGAAGTT JC18 SAT1 flipper GCTCTAGAACTAGTGGATCT JC57 5 NCR of CNA1 AATGATGGAGTTTATGGGAA JC58 5 NCR of CNA1 AACTTCCTCGAGGGGGGGCCGAAAAAAAAAAAGGGGGGGG JC59 3 NCR of CNA1 AGATCCACTAGTTCTAGAGCTTTCTCTTCCTCCTCCTCCTC JC60 3 NCR of CNA1 TGCCCTTTTCTTTGTTTTTG JC61 CNA1 overlap AACAACAATAAGGTGGATGGG JC62 CNA1 overlap TATCTTACCCCTACCTGGTGG JC63 CNA1 ORF GTCAGGAAATACTGTTCAACG JC64 CNA1 ORF CTTCATATAATTGTTCCGA JC48 Disruption confirmation ACAATCAAAGGTGGTCCT JC81 Disruption confirmation AACTTCCTCGAGGGGGGGCC JC82 5 NCR of CNB1 TGTTGTTGGGTAGGTTGCTGT JC83 5 NCR of CNB1 AACTTCCTCGAGGGGGGGCCTTGTTGAAGTTGGATTGGTTG JC86 3 NCR of CNB1 AGATCCACTAGTTCTAGAGCAGCACCTGTAAGATGGTTTAG JC87 3 NCR of CNB1 TGTCCAAAAACGGTTTCCAG JC88 CNB1 overlap TGCCTTTCTGTTCGATTGAT JC89 CNB1 overlap CGACAATAATTGGAATGATGA JC79 CNB1 ORF ATGGGGGCTAACGCAAGTATT JC80 CNB1 ORF TTTAAGGTCAAAGTGTTGGCA JC100 5 NCR of CRZ1 AATATAGAGACGGTGTGTGGG JC101 5 NCR of CRZ1 AACTTCCTCGAGGGGGGGCCGGACAAAATGAAATATAGC JC102 3 NCR of CRZ1 AGATCCACTAGTTCTAGAGCTCAACTTGTTGGTTTGTCTTT JC103 3 NCR of CRZ1 CTAATGTCATGACTTCCCCA JC104 CRZ1 overlap TGAGACATAAAAAGGCGAC JC105 CRZ1 overlap TGATCTCGATATGATTGTCC JC106 CRZ1 ORF TAATCCCTATCCACAGGACGA JC107 CRZ1 ORF ATCACTGGGGGAACAAAACT JC320 CRZ1 complementation AAAAAACCGCGGTCAACTTGTTGGTTTGTCTTT JC321 CRZ1 complementation AAAAAAGAGCTCGTGATGAATCATGAGTCGGA JC288 CRZ1 complementation AAGGTACCAATATAGAGACGGTGTGTGGG JC289 CRZ1 complementation AAAAGCTTAGTAATTTCAACACCACTACT JC292 qpcr ACT1 ORF AGCTCCAGAAGCTTTGTTCAGACC JC293 qpcr ACT1 ORF TGCATACGTTCAGCAATACCTGGG JC345 qpcr CNA1 ORF GCTTCACCTCATCCTTATTGGT JC346 qpcr CNA1 ORF GGTGACACTGGTGTTGTTGTTT JC329 qpcr CNB1 ORF TGAAGAGATCGATAGATTGCG JC330 qpcr CNB1 ORF TTCCCGAGAATGCAGATAACC JC347 qpcr CRZ1 ORF CACAACAATGGCGATGGTAATA JC348 qpcr CRZ1 ORF ACCTTGATGCATGACTTTTCCT JC337 qpcr PMC1 ORF TTGAAACAAGGTGTTGCTGAAG JC338 qpcr PMC1 ORF ATTGTGGTCCTTCCATACATGA JC341 qpcr PMR1 ORF TGAGTACCAGTATCGCTGCATT JC342 qpcr PMR1 ORF GATCACCTGTCGGGTAAGAATC JC362 qpcr CCH1 ORF TGTCACTGATGAAGCTGGCAAA JC363 qpcr CCH1 ORF TGATTAAGCTTAGCCACTGGTGTG JC364 qpcr MID1 ORF TGCCGTAACTATACCTCGATGTTC 1 / 14

2 1 JC365 qpcr MID1 ORF GGGCATAAAAATCCAAAATCAGC Underline indicates sequences complementary to the SAT1 flipper 2 2 / 14

3 Figure S1. C. dubliniensis CNA1, CNB1, and CRZ1 genes were disrupted with the SAT1 flipper. (A) Structure of the CNA1 disruption allele that was made by overlap PCR (see materials and methods for details). PpuMI was used to digest genomic DNAs extracted from specific strains. The PpuMI restriction sites are indicated 5 (-) and 3 (+) of the CNA1 gene. The probe is marked as a bold line. The thick dark arrows represent the FRT sites for FLP recombinase. The thinner arrow on a raised line represents C. albicans MAL2 promoter and the stick with a ball represents C. albicans ACT1 terminator. The Southern blot is shown in the right panel. Sizes of each band were labeled. The strains are WT (CD36), CNA1/cna1::SAT1-FLP (YC31), CNA1/cna1 (YC36), cna1/cna1::sat1-flp (YC40), CNA1/cna1::SAT1-FLP (YC29), CNA1/cna1 (YC73), and cna1/cna1::sat1-flp (YC94). (B) Genomic DNA from CNB1 disruptants was digested with HpaI. The strains are WT (CD36), CNB1/cnb1::SAT1-FLP (YC47), CNB1/cnb1 (YC69), cnb1/cnb1::sat1-flp (YC87), CNB1/cnb1::SAT1-FLP (YC41), CNB1/cnb1 (YC82), and cnb1/cnb1::sat1-flp (YC96). (C) Genomic DNA from CRZ1 disruptants was digested with EcoRV. The strains are WT (CD36), CRZ1/crz1::SAT1-FLP (YC81), CRZ1/crz1 (YC102), crz1/crz1::sat1-flp (YC107), CRZ1/crz1::SAT1-FLP (YC80), CRZ1/crz1 (YC100), and crz1/crz1::sat1-flp (YC108) Figure S2. Complementation by CRZ1 rescues crz1/crz1 mutants in various stresses. (A) Structure of the CRZ1 complementation allele in plasmid pyc393 (see materials and methods for details). EcoRV was used to digest genomic DNA extracted from specific strains. The probe is marked as a red bold line. The Southern blot is shown in the right panel. Sizes of each band are labeled. The strains are WT (CD36), crz1/crz1::sat1-flp (YC108), crz1/crz1 (YC280), and crz1/crz1 + CRZ1 (YC512). (B) Cells were grown overnight in YPD at 30 C, five-fold serially 3 / 14

4 diluted, and spotted onto YPD medium containing SDS, fluconazole (FL), or CaCl 2 at the concentrations indicated. The plates were incubated at 30 C for 48 hr and photographed. (C) Cells were grown overnight and washed twice with ddh 2 O. One hundred microliters containing ~100 cells were spread on Spider media and incubated at 37 C for 9 days. Scale bar=1 mm Figure S3. C. dubliniensis calcineurin is dispensable for growth at elevated temperature. (A) A set of wild type and calcineurin mutants of C. dubliniensis and C. neoformans were grown overnight at 24 C, five-fold serially diluted, and spotted onto YPD to grow for 48 hr at various temperatures. (B) The growth curve of C. dubliniensis wild type and mutant strains at 30 C. Cells were grown overnight at 30 C, washed twice with ddh 2 O, diluted to 0.2 OD/ml in fresh 11 YPD media and incubated at 30 C with shaking at 250 rpm. The OD 600 of cultures were measured at 0, 3, 6, 9, 24, and 30 hr. The experiments were performed in triplicate, and data was plotted using Prism Figure S4. Calcineurin controls increased tolerance to new generation azoles in C. dubliniensis. Cells were grown overnight in YPD at 30 C, five-fold serially diluted, and spotted onto YPD medium ± posaconazole (POS) or voriconazole (VOR) at the indicated concentrations. The plates were incubated at 30 C for 48 hr and photographed Figure S5. crz1/crz1 mutants are hypersensitive to CaCl 2 at elevated temperature in C. dubliniensis and C. albicans. 4 / 14

5 1 Cells were grown overnight in YPD at 30 C, five-fold serially diluted, and spotted onto YPD 2 medium ± CaCl 2 at the indicated concentrations. The plates were incubated at 24 C, 30 C, and 3 37 C for 48 hr and photographed Figure S6. Calcineurin is required for germ tube formation in C. dubliniensis but not in C. albicans. (A) Germ tube formation of wild type and mutant strains in liquid spider medium ± FK506 (1 µg/ml) at 37 C incubated for 2 hr on 96-well polystyrene plates. Arrow heads indicate cells with germ tube formation defects. Scale bar=20 µm. (B) Bar graph showing quantitative percentage of germ tube formation in wild type and mutant strains of C. dubliniensis and C. albicans. At least 200 cells were evaluated in each experiment. The experiments were repeated three times, and error bars represent the mean ± SD. The asterisks represent a statistically significant difference (P<0.05) between wild type and FK506 (1 µg/ml)-treated wild type, cna1/cna1, or cnb1/cnb1 mutants Figure S7. C. dubliniensis is as virulent as C. albicans in a heterologous insect model. Survival of G. mellonella after injection of 10 6 CFU/larva of C. dubliniensis or C. albicans was similar. All infected larva died within 3 days after infection Figure S8. C. dubliniensis exhibits less hyphal growth and damage to oral epithelial cell compared with C. albicans. (A) FaDu oral epithelial cells were incubated with C. dubliniensis or C. albicans for 2, 4, or 6 hr, after which the morphology was examined by differential interference contrast microscopy. Scale bar=10 µm. 5 / 14

6 (B) FaDu oral epithelial cells were incubated with C. dubliniensis or C. albicans for 2, 4, and 6 hr, after which the extent of host cell damage was determined using a 51 Cr release assay. Results are the mean ± SD of three independent experiments. *P<0.05, **P< compared with C. albicans. 6 / 14

7 A 5 3 CNA1-4,000 PpuMI -3,773 5 CNA1 NCR SAT1 FLP MAL2p 1 Kb ACT1t CNA1 1,824 bp PpuMI CNA1 NCR +4,000 Kb Figure S1 cna1::sat1-flp (8.3 kb) CNA1 (6.0 kb) cna1 (4.2 kb) B 5 CNB1 NCR FLP SAT1 MAL2p ACT1t 3 CNB1 NCR 5 3 CNB1 Kb cnb1::sat1-flp (6.5 kb) -4,000 HpaI -1,824 CNB1 522 bp HpaI , CNB1 (2.9 kb) cnb1 (2.4 kb) C MAL2p EcoRV CRZ1-4,000 EcoRV CRZ1 NCR SAT1 FLP EcoRV -243 ACT1t CRZ bp 3 CRZ1 NCR EcoRV +2,236 +4,000 7 / 14 Kb crz1::sat1-flp (5.5 kb) CRZ1 (4.3 kp) crz1 (2.5 kp)

8 A 5 CRZ1 NCR KpnI EcoRV HindIII 1 Kb ACT1t 3 CRZ1 NCR SacII SacI Figure S2 CRZ1 YeGFP RPS10 SAT1 CRZ1-SAT1 (8.7 kb) crz1::sat1-flp (5.5 kb) CRZ1 (4.3 kb) FRT -4,000 FRT +4,000 crz1 (2.5 kb) crz1/crz1 (YC280) EcoRV (+2,236) B WT (CD36) cna1/cna1 (YC40) cnb1/cnb1 (YC87) YPD SDS (0.01 %) FL (10 µg/ml) CaCl 2 (0.3 M) crz1/crz1 YC108 YC280 crz1/crz1 + CRZ1 (YC512) C WT (CD36) crz1/crz1 (YC280) crz1/crz1 + CRZ1 (YC512) Spider, 9 d 8 / 14

9 Figure S3 A C. dubliniensis WT (CD36) cna1/cna1 (YC40) cnb1/cnb1 (YC87) crz1/crz1 (YC108) WT (H99) C. neoformans cna1 (KK1) 24 C 30 C 37 C B 9 / 14

10 Figure S4 C. albicans C. dubliniensis WT (CD36) YC40 cna1/cna1 YC94 YC87 cnb1/cnb1 YC96 YC107 crz1/crz1 YC108 WT (SC5314) cna1/cna1 (SCCMP1M4) Ref WT (DAY185) cnb1/cnb1 (JRB64) crz1/crz1 (OCC1.1) YPD POS (0.1 µg/ml) VOR (0.1 µg/ml) 10 / 14

11 Figure S5 C. dubliniensis WT (CD36) cna1/cna1 (YC40) cna1/cna1 (YC94) cnb1/cnb1 (YC87) cnb1/cnb1 (YC96) crz1/crz1 (YC107) crz1/crz1 (YC108) crz1/crz1 + CRZ1 (YC512) C. albicans WT (DAY185) cnb1/cnb1 (JRB64) cnb1/cnb1 + CNB1 (MCC85) crz1/crz1 (OCC1.1) crz1/crz1 + CRZ1 (OCC7) WT (CAF2-1) cna1/cna1 (DSY2091) cna1/cna1 + CNA1 (DSY2115) crz1/crz1 (DSY2195) crz1/crz1 + CRZ1 (MKY268) YPD CaCl 2 (0.4 M) CaCl 2 (0.3 M) CaCl 2 (0.2 M) 37 C 30 C 24 C 11 / 14

12 Figure S6 A C. dubliniensis +FK506 WT (CD36) cna1/cna1 (YC40) cnb1/cnb1 (YC87) crz1/crz1 (YC108) C. albicans +FK506 WT (SC5314) cna1/cna1 (SCCMP1M4) cna1/cna1 +CNA1 (SCCMP1MK2) B C. albicans C. dubliniensis WT (CD36) cna1/cna1 (YC40) cnb1/cnb1 (YC87) crz1/crz1 (YC108) WT (SC5314) cna1/cna1 (SCCMP1M4) cna1/cna1+cna1 (SCCMP1MK2) FK * * * Germ tube formation (%) 12 / 14

13 13 / 14 Figure S7

14 Figure S8 A 2 h 4 h 6 h C. dubliniensis (CD36) C. albicans (SC5314) B * ** ** 14 / 14